Four cDNAs for spermidine synthase (SPDS), which converts the diamine putrescine to the higher polyamine spermidine using decarboxylated S-adenosylmethionine as the co-factor, were isolated from Nicotiana sylvestris, Hyoscyamus niger, and Arabidopsis thaliana. When the N.sylvestris SPDS cDNA was expressed in a SPDS-deficient E. coli mutant, the recombinant protein showed high SPDS activity, but did not have any spermine synthase activity. The plant SPDSs have molecular masses of about 34 kDa, possess the co-factor binding motifs which have been proposed for S-adenosylmethionine, and are more homologous in amino acid sequence to tobacco putrescine N-methyltransferase (PMT) than to SPDSs from mammals and E. coli. The SPDS gene is expressed in root, stem, and leaf in N.sylvestris, whereas the PMT gene is expressed only in root. The potential evolution of plant SPDS and PMT, and their evolutionary relationships with animal SPDS are discussed.
The putrescine N-methyltransferase (PMT) cDNA clone previously isolated from tobacco encodes a spermidine synthase-like protein with an 11 amino acid element repeated four times in tandem at the amino terminus. Genomic Southern blot analyses indicated that this N-terminal repeat array is found in tobacco PMTs but absent in Hyoscyamus and Atropa PMTs. A truncated tobacco PMT in which this repeat array was entirely removed still retained full enzymatic activity when expressed in Escherichia coli. Three PMT genes (NsPMT1, NsPMT2, NsPMT3) isolated from Nicotiana sylvestris encode two, five, and nine tandem repeats, respectively, in the first exon, but otherwise encode highly conserved proteins. Analysis of PCR fragments amplified from the genomes of N. tabacum and its two probable progenitors shows that one of the nine repeat elements in NsPMT3 was precisely deleted in the corresponding N. tabacum gene. These results indicate that direct tandem repeats of a 33 bp sequence that encodes 11 amino acids of no obvious function were added to the ancestral Nicotiana PMT gene, and that the tandem repetition was genetically very unstable, contracting or expanding during evolution of the Nicotiana species.
A cDNA clone of a novel cytochrome P450, CYP76A4, was isolated from Petunia hybrida. The cDNA clone contained an open reading frame (ORF) encoding a predicted 510 amino acid polypeptide. The CYP76A4 cDNA was expressed in yeast Saccharomyces cerevisiae AH22. Recombinant yeast microsomes containing the CYP76A4 hemoprotein were found to catalyze (!-1)-hydroxylation of lauric acid.
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